Molecular and General Genetics MGG

, Volume 196, Issue 2, pp 339–344 | Cite as

The transformation of mycelial spheroplasts of Neurospora crassa and the attempted isolation of an autonomous replicator

  • F. P. Buxton
  • A. Radford


A new transformation protocol for mycelially derived spheroplasts of Neurospora was developed and optimised for a number of components. This protocol was used to screen a bank of 700 clones for high frequency transforming plasmids using miniscreen DNA. Four with significantly increased frequency were found, but they were not capable of autonomous replication. We discuss the conditions that an autonomously replicating plasmid must meet in order to exist in a mycelial fungus such as Neurospora.


Mycelial Fungus Frequency Transforming Neurospora Crassa Transformation Protocol Transforming Plasmid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Alton NK, Hautala JA, Giles NH, Kushner SR, Vapnek D (1978) Transcription and translation in Escherichia coli of hybrid plasmids containing the catabolic dihydroquinate dehydrogenase gene from Neurospora crassa. Gene 4:241–259Google Scholar
  2. Buxton FP, Radford A (1983) Cloning of the structural gene for orotidine 5′-phosphate carboxylase of Neurospora crassa by expression in Escherichia coli. Mol Gen Genet 190:403–405Google Scholar
  3. Case ME (1982) Transformation of Neurospora crassa utilising recombinant plasmid DNA. In: Hollaender A, DeMoss RD, Kaplan S, Konisky J, Savage D, Wolie RS (eds) Genetic Engineering of Microorganisms for Chemicals. Plenum Publishing Corporation, New York, London, pp 87–100Google Scholar
  4. Case ME, Schweizer M, Kushner SR, Giles NH (1979) Efficient transformation of Neurospora crassa by utilising hybrid plasmid DNA. Proc Natl Acad Sci USA 76:5259–5263Google Scholar
  5. Chan CSM, Tye BK (1980) Autonomous replication sequences in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 77:6329–6333Google Scholar
  6. Clarke L, Carbon J (1980) Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature 287:504–509Google Scholar
  7. Dhawale SS, Paietta JV, Marzluff GA (1984) A new rapid and efficient transformation procedure for Neurospora. Curr Genet 8:77–79Google Scholar
  8. Emerson S (1963) Slime a plasmodioid variant of Neurospora crassa. Genetica 34:162–182Google Scholar
  9. Hyman BC, Cramer JH, Rownd RH (1982) Properties of Saccharomyces cerevisiae mitochondrial DNA segment confering high frequency yeast transformation. Proc Natl Acad Sci USA 79:1578–1582Google Scholar
  10. Ish-Horowicz D, Burke JF (1981) Rapid and efficient cosmid cloning. Nucl Acid Res 9:2989–2998Google Scholar
  11. Kearsey S (1983) Analysis of sequences conferring autonomous replication on baker's yeast. EMBO J 2:1571–1575Google Scholar
  12. Kinnaird JH, Keighren MA, Kinsey JA, Eaton M, Fincham JRS (1982) Cloning of the am (glutamate dehydrogenase) gene of Neurospora crassa through the use of a synthetic DNA probe. Gene 20:387–396Google Scholar
  13. Muller HJ (1932) Some genetic aspects of sex. Am Natur 66:118–138Google Scholar
  14. Muller HJ (1964) The relation of recombination to mutational advance. Mutat Res 1:2–9Google Scholar
  15. Murray AW, Szostack JW (1983) Pedigree analysis of plasmid segregation in yeast. Cell 34:961–970Google Scholar
  16. Perkins DD, Barry EG (1977) The cytogenetics of Neurospora crassa. Adv Genet 19:133–285Google Scholar
  17. Pittenger TH, Kimball AW, Atwood KC (1955) Control of nuclear ratios of Neurospora heterokaryons. Am J Botany 42:954–958Google Scholar
  18. Radford A, Pope S, Sazci A, Fraser MJ, Parish JH (1981) Liposome mediated genetic transformation of Neurospora crassa. Mol Gen Genet 184:567–569Google Scholar
  19. Robertson NF (1961) Mycology in contemporary botanical thought. MacLeod AM, Cobley LS (eds). Oliver & Boyd, Edinburgh and London, pp 133–148Google Scholar
  20. Schechtman MG, Yanofsky C (1983) Structure of the trifunctional trp-1 gene from Neurospora crassa and its aberrant expression in Escherichia coli. J Mol Appl Genet 2:83–89Google Scholar
  21. Selitrenikoff CP, Lilley BL, Zucker R (1981) Formation and regeneration of protoplasts derived from a temperature sensitive osmotic strain of Neurospora crassa. Exp Mycol 5:155–161Google Scholar
  22. Stinchcomb DT, Struhl K, Davis RW (1979) Isolation and characterisation of a yeast chromosomal replicator. Nature 282:39–43Google Scholar
  23. Stohl LL, Lambowitz AM (1983) Construction of a shuttle vector for the filamentous fungi Neurospora crassa. Proc Natl Acad Sci USA 80:1058–1062Google Scholar
  24. Wiley WR (1974) Isolation of spheroplasts and membrane vesicles from yeast and filamentous fungi. Methods Enzymol 31:609–626Google Scholar

Copyright information

© Springer-Verlag 1984

Authors and Affiliations

  • F. P. Buxton
    • 1
  • A. Radford
    • 1
  1. 1.Department of GeneticsUniversity of LeedsLeedsEngland

Personalised recommendations